Abstract
The metabolism, excretion, and pharmacokinetics of 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (INCB018424), a potent, selective inhibitor of Janus tyrosine kinase1/2 and the first investigational drug of its class in phase III studies for the treatment of myelofibrosis, were investigated in healthy human subjects given a single oral 25-mg dose of [14C]INCB018424 as an oral solution. INCB018424 and total radioactivity were absorbed rapidly (mean time to reach the maximal drug concentration <1 h), declining in a monophasic or biphasic fashion (mean t1/2 of 2.32 and 5.81 h, respectively). Recovery of administered radioactivity was fairly rapid (>70% within 24 h postdose) with 74 and 22% recovered in urine and feces, respectively. Parent compound was the predominant entity in the circulation, representing 58 to 74% of the total radioactivity up to 6 h postdose, indicating that the overall circulating metabolite burden was low (<50% of parent). Two metabolite peaks in plasma (M18 and a peak containing M16/M27, both hydroxylations on the cyclopentyl moiety) were identified as major (30 and 14% of parent based on area under the curve from 0 to 24 h). The exposures of other circulating INCB018424-related peaks were <10% of parent, consisting of mono- and dihydroxylated metabolites. The profiles in urine and feces consisted of hydroxyl and oxo metabolites and subsequent glucuronide conjugates with parent drug accounting for <1% of the excreted dose, strongly suggesting that after an oral dose, INCB018424 was >95% absorbed. In healthy subjects administered daily oral doses of unlabeled INCB018424, there were minimal differences in parent and metabolite concentrations between day 1 and day 10, indicating a lack of accumulation of parent or metabolites between single and multiple dosing.
Introduction
The Janus kinase family of protein tyrosine kinases (JAKs) plays an important role in the signaling of a number of cytokines and hematopoietic growth factors (Ghoreschi et al., 2009). Multiple cytokines, including those in the interleukin-2 and interleukin-6 family, signal via JAK1 and JAK3 enzymes to exert profound effects on lymphocyte differentiation, survival, and function (Nelson and Willerford, 1998; Vainchenker et al., 2008). In addition, JAK2 plays a central role in the signal transduction of erythropoietin and thrombopoietin (Neubauer et al., 1998; Parganas et al., 1998) and has been shown to play a critical role in erythropoiesis and thrombopoiesis in humans. 3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (INCB018424) is an orally active and potent inhibitor of JAKs with greater than 100-fold selectivity against a panel of 26 non-JAK kinases and is in development for the treatment of myeloproliferative neoplasms (Lin et al., 2009; Quintás-Cardama et al., 2010). INCB018424 has demonstrated efficacy in myelofibrosis, the most serious of the chronic myeloproliferative neoplasms for which there is no approved therapy (Verstovsek et al., 2009a). Data from ongoing studies in myelofibrosis demonstrate marked reductions in spleen size and improvements in constitutional symptoms, which are maintained over many months in subjects with continuous therapy with oral administration of 10 to 25 mg b.i.d. INCB018424 (Mesa and Tefferi, 2009; Verstovsek et al., 2009b).
INCB018424 exhibits rapid oral absorption and dose proportional systemic exposures. A manuscript submitted for publication (J. Shi, X. Chen, T. Emm, P. Scherle, R. McGee, R. Landman, Y. Lo, E. McKeever, N. Punwani, W. Williams, et al.) demonstrated that INCB018424 had moderate clearance and volume of distribution, with an approximate 3-h terminal-phase elimination half-life in plasma. Accumulation of INCB018424 after multiple dose administration was negligible. Nonclinical metabolism studies in mice, rats, and dogs administered oral doses of [14C]INCB018424 indicated rapid excretion of drug-derived radioactivity by both urinary and fecal routes with unchanged parent accounting for <20% of the excreted dose, indicating extensive metabolism (A. Shilling and F. Nedza, unpublished data). Species differences were minor with circulating and excreted metabolites mainly consisting of hydroxylation(s), oxo, and in some cases, subsequent O-glucuronides. In this study, the excretion, mass balance, metabolism, and pharmacokinetics of [14C]INCB018424 after a single oral 25 mg (90 μCi) dose were evaluated in healthy human subjects. The metabolite profiles in plasma, urine, and feces were determined to identify and quantitate metabolites as well as the routes of excretion for INCB018424 and its metabolites. The data generated from these studies can be used to support the safety evaluation of INCB018424 and its metabolites by a comparison with the corresponding data generated in nonclinical species. It is essential to ensure that the nonclinical species used in toxicological assessments have been exposed to major human metabolites at concentrations that approach or exceed those observed in humans at the highest proposed clinical dose.
Materials and Methods
Chemicals.
[14C]INCB018424 was synthesized as the phosphate salt by PerkinElmer Life and Analytical Sciences (Waltham, MA) with a specific activity for the stock powder of 59.98 mCi/mmol and stored at −70°C until use. The radiolabeled material was then added to bottles containing INCB018424 phosphate drug substance to achieve a concentration of approximately 25 mg (90 μCi) and stored ambient at 15 to 30°C (59–86°F). INCB018424 phosphate drug substance has been shown to be stable for at least 6 months at accelerated conditions of 40°C/75% relative humidity and 9 months at 25°C/60% relative humidity. The radiochemical purity as determined by high-performance liquid chromatography (HPLC) was 99.18%. The internal standard was a stable isotopically labeled INCB018424 containing four 13C on the pyrrolopyrimidine. The metabolite reference standards (M7, M8, M9, M11, M16, M18, M27, and M49) were synthesized at Incyte Corporation (Wilmington, DE) and characterized by NMR. Control plasma used to prepare the calibration standards, quality control samples, and blank samples and to perform dilutions containing potassium EDTA as an anticoagulant with 0.02% ascorbic acid were purchased from Bioreclamation (Hicksville, NY). All other chemicals were of reagent grade or better.
Clinical Study Conduct.
The single-dose 14C study (protocol INCB 18424-134) and a 10-day multiple dose study with unlabeled compound (protocol INCB 18424-132) were conducted at Quintiles Phase I Services (Overland Park, KS). Both studies were conducted in full accordance with the guidelines of Declaration of Helsinki, Good Clinical Practice, and local laws regarding the protection of the rights and welfare of human participants in biomedical research. The clinical study protocol, amendments, informed consent documents(s), and any other appropriate study-related documents were reviewed and approved by an independent ethics committee/institutional review board. Informed consent for all participants was obtained before the conduct of any study-related procedures.
Subjects, Dose Preparation, Administration, and Sample Processing for Single-Dose Study with [14C]INCB018424.
For the 14C study, subjects were admitted to the facility the day before dosing and remained in the clinic for 5 days or until less than 1% of the dose was recovered in two subsequent urine/fecal samples or ≥90% of the radiolabeled dose was recovered. Subjects were assessed for continued study eligibility, which included review of prior medications, intercurrent illness assessment, targeted physical examination, measurement of vital signs, 12-lead ECG, and collection of samples for clinical laboratory assessments (hematology, serum chemistry, urinalysis, and drug screen including alcohol). Subjects remained confined to the clinic and fasted overnight for at least 10 h. Six healthy adult male volunteers, ages 19 to 37 years, and weighing 57 to 89 kg with a body mass index between 20 and 29 kg/m2, were given a single oral dose of approximately 25 mg (90 μCi) of [14C]INCB018424 as an oral solution. All subjects who participated in the study received daily doses of FiberCon (active ingredient 625 mg of calcium polycarbophil, equivalent to 500 mg of polycarbophil) in the evening beginning on day 1 as prophylaxis. On days 2 through 5 and before discharge, study procedures included the measurement of vital signs every 24 h, targeted physical examinations, collection of samples for clinical laboratory assessments (hematology, serum chemistry, and urinalysis) on day 4, review of concomitant medications, and adverse event assessments. There were no subjects who received treatment for adverse events and no other reported previous or concomitant medications. [14C]INCB018424 drug substance was reconstituted in sterile water to make a solution before dosing. Subjects received a single dose of 25 mg (90 μCi) of [14C]INCB018424 as an oral solution in purified water (59 ml) on day 1 followed by 240 ml of water after an overnight fast of 10 h. A 1-ml aliquot of the dosing solution for each subject was collected to determine the radioactivity concentration by liquid scintillation counting (LSC) and assess radiopurity and chemical purity of the dosing solution before and after dosing using HPLC in-line radiodetection. The radiochemical purity of the dose solutions was >98.7% for all aliquots and similar for predose and postdose samples (mean of 99.0 and 99.2%, respectively). Subjects remained fasting for 1 h postdose at which time a standard, dietician approved meal, was served.
Blood samples were collected into Vacutainer evacuated collection tubes containing potassium EDTA at designated time points [pharmacokinetics (4 ml): 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, and 36 h; metabolite profiling (20 ml): 0, 1, 2, 6, 12, 24, and 36 h]. Blood samples were gently mixed by slowly inverting the collection tube several times and centrifuged at approximately 2000g for 15 min at approximately 5°C. For urine, individual voiding samples for each subject were pooled into containers (polypropylene or polyethylene) for the designated time intervals and stored at 5°C during the collection period. At the end of each collection time interval, samples were thoroughly mixed by vortexing. Feces samples were collected at 5°C, put into containers for designated time intervals, and homogenized with approximately two times their weight of water. Plasma, blood cells, urine, and feces were stored at −20 to −80°C.
Subjects, Dose Preparation, Administration, and Sample Processing for Multiple Dose (10-Day) Study with Unlabeled INCB018424.
Study INCB 18424-132 was a randomized, double-blind, placebo-controlled, rising multiple-dose tolerability and pharmacokinetic study. In brief, nine healthy subjects (seven male and two female) were administered 50 mg of unlabeled INCB018424 (two 25-mg capsules; size 0, crystalline, no excipients) orally every 12 h (morning and evening) for 10 days beginning with the morning dose on day 1 through the morning dose on day 10. All morning doses were preceded by at least an 8-h fast from food and a 1-h fast from water and were followed by a fast from food and water for at least 1 h postdose. All evening doses were preceded by at least a 3-h fast from food and a 1-h fast from water and were followed by a fast from food and water for at least 1 h postdose. Venous blood samples (4 ml) were collected 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after the first dose on day 1 and at these time points in addition to samples at 16, 24, 36, and 48 h after the last dose (morning) on day 10 to measure plasma concentrations of INCB018424. Daily study procedures included the measurement of vital signs, targeted physical examinations, collection of samples for clinical laboratory assessments (hematology, serum chemistry, and urinalysis), review of concomitant medications, and adverse event assessments.
Determination of Radioactivity.
Plasma radioactivity was determined by direct liquid scintillation counting, and blood cell radioactivity was determined in duplicate by combustion with the resulting 14CO2 assayed by LSC. Blank plasma and blank blood cells purchased from Bioreclamation, Inc. were used as background subtraction in the radioanalysis as well as for matrix radioactivity recovery checking during the radioanalysis. For urine, duplicate aliquots were taken to determine the total radioactivity by direct LSC. A predose urine sample prepared as a pool from all six subjects' predose urine samples was used as a blank matrix for background subtraction and blank matrix radioactivity recovery determination. The radioactivity of fecal homogenate samples (0.5 g) was determined in duplicate by combustion with the resulting 14CO2 assayed by LSC. A pooled predose fecal homogenate sample prepared by taking predose fecal homogenate samples from three subjects (002, 004, and 005) was used as blank matrix for background subtraction and matrix radioactivity recovery tests.
Metabolite Profiling and Characterization.
For the 14C study, plasma samples were pooled equally by time point for the six subjects at predose, 1, 2, 6, 12, 24, and 36 h and by subject (approximately 1.5 ml/time point). For sample preparation and analyses, an aliquot of each pooled plasma sample was taken for LSC to determine the initial radioactivity. To each pooled plasma sample, 18 ml of 1% formic acid in acetonitrile was added and centrifuged at 2000g at 4°C for 10 min. The supernatant was harvested and passed through a preconditioned C-18 SPE column (Sep-Pak, 6 ml; Waters, Milford, MA). Formic acid (1%) in acetonitrile was added to the pellet, vortexed, and centrifuged at 2000g and 4°C for 10 min. The supernatant was passed through the SPE column. The extraction was repeated two more times with 1% formic acid in acetonitrile-H2O (v/v, 90:10), and the SPE column was rinsed with formic acid in methanol. The elute was concentrated under an N2 stream at 30°C using a Zymark concentrator and centrifuged at 18,000g for 20 min. Duplicate 25-μl aliquots were taken to determine the radioactivity in the final extract and to assess the final recovery of radioactivity from the extracted radioactivity concentration procedure. Subsamples of equal amounts of urine from each of the six subjects were pooled within time intervals 0 to 8 h, 8 to 24 h, and 24 to 48 h, and each pooled urine sample was centrifuged at 2000g for 10 min. Subsamples of fecal homogenates from 24 to 48 h, 48 to 72 h, and 72 to 96 h collection from at least three of the six subjects were pooled for each of these collection intervals for metabolite profiling, vortexed, and centrifuged at 2000g and 4°C for 10 min. The supernatant was harvested and passed through a preconditioned C-18 SPE column, and the pellet was extracted as described for plasma.
Samples were assayed using electrospray ionization in the positive scan mode with an API 4000 Qtrap LC-MS/MS system (Applied Biosystems, Foster City, CA) at room temperature. The mass spectrometer was coupled with an Agilent 1100 series pump (Agilent Technologies, Santa Clara, CA) and LEAP CTC-PAL autosampler (LEAP Technologies, Carrboro, NC) using a Symmetry C-18 HPLC column (4.6 × 250 mm; Waters). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B of 1:1 methanol-acetonitrile with a flow rate of 1.0 ml/min. The gradient started as 10% B from 0 to 45 min, 20% B from 45 to 50 min, 50% B from 50 to 55 min, 90% B from 55 to 57.1 min, and 10% B from 57.1 to 65 min. For selected samples, an extended gradient was used for plasma (10% B from 0 to 45 min, 20% B from 45 to 90 min, 40% B from 90 to 92 min, 90% B from 92 to 92.1 min, and 10% B from 92.1 to 97 min) and urine and feces (10% B from 0 to 45 min, 20% B from 45 to 90 min, 40% B from 90 to 92 min, 90% B from 92 to 97.1 min, and 10% B from 97.1 to 102 min). The high-performance liquid chromatograph was coupled to a Ramona-90 radiochemical detector (Raytest, Straubenhardt, Germany) with a 500-μl liquid scintillation cell using Ultima Gold scintillation fluid. For metabolite identification, selected samples were assayed using electrospray ionization in the positive ion scan mode using multiple reaction monitoring (MRM) and MS/MS fragmentation with an API 4000 Qtrap LC-MS/MS system coupled to an Agilent 1100 series pump and LEAP CTC-PAL autosampler using the same column and under HPLC conditions similar to those described for metabolite profiling. Product ion scans to confirm the MS/MS fragmentation of metabolites compared with reference standards was also performed when applicable.
For the unlabeled 10-day multiple dose study (50 mg b.i.d.), residual plasma samples for each subject were pooled according to the Hamilton pooling method (Hamilton et al., 1981) for predose, day 1, and day 10. Aliquots of the pooled plasma samples (150 μl) were precipitated with 2 volumes of acetonitrile, vortex-mixed, and centrifuged. The supernatants were analyzed using electrospray ionization LC-mass spectrometry with a Thermo Finnigan LCQ Deca-XP Plus ion-trap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA) in positive ionization mode. The mass spectrometer was coupled to a Shimadzu Sil HT-C combined autosampler/controller linked to with a Shimadzu LC-10A binary gradient pump system (Shimadzu Scientific Instruments, Columbia, MD). Separation of INCB018424 and its metabolites was achieved using a Zorbax XDB C-18 HPLC column (3.0 × 150 mm, 3.5 μm; Agilent Technologies). Mobile phase A consisted of 5 mM ammonium formate in Millipore water adjusted to pH 3.2 with formic acid, and mobile phase B consisted of 90% acetonitrile-10% methanol with a flow rate of 300 μl/min. Gradient started as 10% B from 0 to 45 min, 40% B from 45 to 50 min, 90% B from 50 to 54.1 min, and 10% B from 54.1 to 60 min.
Quantitation of INCB018424 by LC-MS/MS.
For quantitation of INCB018424 for pharmacokinetics determination from both clinical studies, the plasma samples were assayed by a validated LC-MS/MS method in compliance with the US Food and Drug Administration Good Laboratory Practice regulations, 21 CFR 58, with a linear range of 1 to 1000 nM. For each sample, 50 μl of plasma was extracted by liquid-liquid extraction using methyl t-butyl ether with the internal standard (stable isotopically labeled INCB018424 containing four 13C on the pyrrolopyrimidine). The extract was injected onto a HPLC system consisting of Agilent 1100 series pumps and a LEAP HTC PAL autosampler. INCB018424 and the internal standard were chromatographed on a Synergi Polar-RP 80A column (30 × 2.0 mm, 4 μ; Phenomenex, Torrance, CA) under isocratic conditions containing 55% acetonitrile and 45% 2 mM ammonium acetate with a flow rate of 300 μl/min. The HPLC effluent was monitored on a API-4000 liquid chromatography-tandem mass spectrometer (Applied Biosystems) with TurboIonSpray ionization (electrospray) in positive ion mode and MRM detection. Positive ions were detected by MRM with precursor→product ion pairs of 307.3→186.3 atomic mass units for INCB018424 and of 311.3→190.3 atomic mass units for the internal standard. The peak areas of analyte and internal standard and concentrations of the analyte were calculated by Analyst (version 1.4.1; Applied Biosystems). The calibration curves were obtained by a weighted (1/x2) least-squares linear regression analysis. The concentration of INCB018424 (nanomolar) for each sample was interpolated using the measured peak area ratios of analyte to internal standard.
Pharmacokinetic Analysis.
All blood samples were collected within 10 min of the scheduled time (within 5 min for all time points between 0 and 12 h postdose), and therefore the schedule times relative to the time of dose administration were used for all pharmacokinetic analyses. Standard noncompartmental pharmacokinetic methods were used to analyze the INCB018424 and total radioactivity plasma concentration data using WinNonlin (version 5.0.1; Pharsight Corporation, Mountain View, CA). The maximal drug plasma concentration (Cmax) and time to reach the maximal drug concentration (Tmax) values were taken directly from the observed plasma concentration data. The terminal-phase disposition rate constant (λz) was estimated using a log-linear regression of the concentration data in the terminal disposition phase, and t1/2 was estimated to be ln(2)/λz. The plasma AUC0–t was estimated using the linear trapezoidal rule for increasing concentrations and the log-trapezoidal rule for decreasing concentrations, and the total AUC0–∞ was calculated as AUC0–t + Ct/λz.
Results
Pharmacokinetics.
The pharmacokinetics of total radioactivity and INCB018424 in plasma of healthy subjects after a single oral dose of 25 mg (90 μCi) of [14C]INCB018424 are shown in Table 1. The corresponding plasma concentration-time curves are shown in Fig. 1. INCB018424-derived radioactivity was rapidly absorbed in blood cells and plasma, attaining peak plasma concentrations within 2 h after administration. The mean Cmax and AUC0–∞ values for total radioactivity were 1355 and 6631 nM Eq · h, respectively, with a range for the six subjects of 910 to 2874 and 5660 to 10,629 nM Eq · h, respectively. The parent drug plasma concentration profile also showed a rapid absorption phase, attaining peak concentrations within 1 h after administration and declining in a monophasic or biphasic fashion with a mean observed terminal half-life of 2.32 h. The mean Cmax and AUC0–∞ values for INCB018424 were 1093 nM and 3200 nM · h, respectively, with a range for the six subjects of 609 to 2387 nM and 2206 to 5911 nM · h, respectively. The mean ratio of AUC0–∞ for INCB018424 compared with total radioactivity was 0.47 (range of 0.39–0.56), suggesting that INCB018424 represented approximately half of the INCB018424-related equivalents in plasma.
Excretion of Radioactivity.
The excretion of INCB018424-derived radioactivity in urine and feces of healthy human subjects administered an oral dose of [14C]INCB018424 [25 mg (90 μCi)] is shown in Table 2. The total mean recovery of the administered radioactivity was 96% with a mean of 74 and 22% in urine and feces, respectively, indicating that urine was the major route of excretion for INCB018424-derived radioactivity. In general, the excretion patterns were similar between individual subjects with greater than 70% of dosed radioactivity excreted within 24 h, except for subject 002 whose recovery was 50%. Subject 002 had a lower amount of urine from 0 to 8 h (159 g) compared with that for subjects 001, 003, 004, 005, and 006 (446, 999, 584, 522, and 993 g, respectively; data not shown) due to loss of an unspecified amount of urine in a shower drain. This resulted in a lower total recovery for subject 002 (86%) compared with those for the other five subjects for whom total recovery was ≥94%. An additional 3 to 21 and 0.8 to 13% was excreted within 24 to 48 h and 48 to 72 h postdose, respectively. The average total excretion was <8% beyond 72 h postdose.
Metabolite Profiling in Plasma.
The metabolic profiles in plasma determined in healthy human subjects administered a single oral 25 mg (90 μCi) dose of [14C]INCB018424 are shown in Tables 3 and 4. The chemical structures of INCB018424-derived metabolites and proposed metabolic pathways are shown in Fig. 2. Representative radiochromatograms for plasma are shown in Fig. 3. Plasma profiles were similar qualitatively and quantitatively for samples pooled by time point (Table 3) or by subject (Table 4). For plasma pooled by time point, a total of 10 peaks were identified, accounting for 97% of the total radioactivity in plasma. INCB018424 (m/z 307) was the predominant peak present in plasma representing 74, 66, and 58% of the total radioactivity 1, 2, and 6 h postdose, respectively. The largest metabolite peak was M18 (m/z 323), corresponding to a 2-hydroxylation on the cyclopentyl moiety, which represented 7.3, 9.1, and 14% of the radioactivity at 1, 2, and 6 h postdose. At 12 h, parent drug represented a smaller proportion of the total radioactivity compared with M18 (25 and 52%, respectively), albeit the total radioactivity was ∼10% of Cmax. Individual peaks could not be characterized in plasma samples beyond 12 h postdose because of low radioactivity. Based on the extrapolated area under the curve from 0 to 24 h (AUC0–24), there were two major metabolite peaks in human plasma, M18 (2-hydroxycyclopentyl) and a coeluting peak consisting of two stereoisomers, M16 and M27 (3-hydroxycyclopentyl). The AUC0–24 values in plasma for these two peaks were 17 and 8.3%, respectively, of the total radioactivity and 30 and 14%, respectively, of the AUC0–24 for INCB018424. Seven other peaks observed in plasma listed in descending order each had AUC0–24 values that were <10% of INCB018424 levels: M8 (m/z 323; 3-hydroxylation of cyclopentyl moiety), M11 (m/z 321; 3-oxocyclopentyl), M7 (m/z 323; 3-hydroxycyclopentyl), M38 (m/z 339; bishydroxycyclopentylpyrrolopyrimidine), M49 (m/z 339; a keto alcohol on the pyrrolopyrimidine moiety, M9 (m/z 321; 3-oxocyclopentyl), and M27 (m/z 323; 3-hydroxycyclopentyl).
For samples pooled by subject, the metabolite profiles showed only minor interindividual differences and in all six subjects INCB018424 was the predominant peak representing a mean of 63% of the total radioactivity (range 52–67%). M18 was the only metabolite that represented >10% of the radioactivity in plasma (mean 12%; range 7–17%). The mean radioactivity for all other metabolites observed in plasma, listed in descending order (M16/M27, M8, M11, M7, M49, M9, and M38) were <5% of total. Some of these minor metabolites were not observed in all subjects.
In the multiple dose study with unlabeled compound, the metabolites observed, peak area ratios of these metabolites, and the relative percentage of metabolites compared with INCB018424 did not change between day 1 and day 10 (Table 5). The terminal half-life for INCB018424 was similar on day 1 and day 10 of this study (3.1 and 3.2 h, respectively). M18 was the only metabolite that was >10% of parent (16.5 and 12.6% of INCB018424 on day 1 and day 10, respectively, compared with 30% based on data from the 14C study). There did not appear to be a gender difference although the sample size was too small (seven males and two females) to draw any definitive conclusions.
Metabolite Profiling in Urine and Feces.
The metabolic profiles in urine and feces collected between 0 to 48 h and 24 to 96 h, respectively, are shown in Table 6. Radioactivity in urine samples beyond 48 h postdose and in feces samples at 0 to 24 h and beyond 96 h postdose were too low to identify any individual peaks. Unchanged parent represented <1% of the administered dose in urine and feces, indicating the primary elimination pathway for INCB018424 after oral dosing is by metabolic clearance. Representative radiochromatograms for urine and feces are shown in Fig. 3. In urine, the two largest peaks contained M16/M27, representing approximately 19, 25, and 25% of the radioactivity (by radio-HPLC) at 0 to 8 h, 8 to 24 h, and 24 to 48 h postdose, respectively, and M11, representing 24 and 20% of the radioactivity at 0 to 8 h and 8 to 24 h postdose (not observed after 24 h). These metabolites each accounted for 15 to 16% of the administered dose between 0 and 48 h. M7, M8, and M49 each contributed 11 to 18% of radioactivity and 8.0 to 11.0% of the dose. All other peaks, consisting of mono- and dihydroxylations (m/z 323 and 339, respectively) as well as O-glucuronides (m/z 499), represented <7% of the radioactivity and <3% of the administered dose.
In feces, M8 was the largest INCB018424-related component, representing approximately 22, 24, and 29% of the radioactivity by HPLC in the samples at 24 to 48 h, 48 to 72 h, and 72 to 96 h postdose, respectively, accounting for approximately 2% of the administered dose at each time interval. M7, M16/M27, and M49 were also significant metabolites, each representing between 10 and 16% of the radioactivity and approximately 2.3 to 4.6% of the administered dose between 24 and 96 h postdose. Other peaks in feces, which each represented <10% of the radioactivity and <2% of the administered dose were M43 (m/z 339) and M45 (m/z 339), both dihydroxylations of the cyclopentylpropanenitrile moiety, and M18 and M31 (m/z 323), both hydroxylations on the 2-position of the cyclopentyl moiety.
Discussion
The mass balance excretion of INCB018424 after a single dose and the metabolism of INCB018424 after single and multiple dosing was characterized in humans. After a single oral dose of [14C]INCB018424 to healthy human subjects, excretion of total radioactivity was fairly rapid with a mean of >70% of the dose recovered by 24 h postdose. The primary route of excretion for total radioactivity was urine with a smaller proportion recovered in feces. INCB018424 was extensively metabolized in humans (<1% eliminated as parent drug), whereas the primary component in circulation was unchanged parent, which accounted for approximately half of the total INCB018424-related material in plasma.
Understanding the biopharmaceutical characteristics of a new chemical entity is an integral part of drug development. Amidon et al. (1995) have proposed a biopharmaceutical classification system that bins drugs into four classes based on solubility and dissolution and permeability and absorption. The requirement for demonstrated bioequivalence for the marketed product to that used in registrational studies can be waived for compounds with high solubility and high permeability (considered class I). Although solubility can be readily documented using conventional saturation solubility studies, demonstration of high permeability requires extensive in vitro characterization including validation of the assay system used and other factors. An alternative is the extent of absorption estimated from human pharmacokinetic studies. According to the Food and Drug Administration Guidance for Industry (Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System 2000; http://www.fda.gov/cder/guidance/index.htm), a drug substance is considered highly permeable when the extent of absorption in humans is determined to be 90% or more of an administered dose based on mass balance determination. In the case of INCB018424, the drug substance is considered highly soluble because the highest dosage strength (25 mg) was soluble in water at a volume of less than 250 ml over the pH range of 1.0 to 7.5. In the current study, total recovery of administered radioactivity was 96% (74% in urine and 22% in feces) with <1% of the recovered dose excreted as parent drug, indicating that >99% of the administered dose was metabolized. Because metabolites of a compound that is stable in intestinal fluids must originate from absorbed drug (as indicated by Benet et al., 2008), results from this study strongly suggest that after an oral dose, INCB018424 was >95% absorbed (>99% of 96% recovered). Hence, these results provide crucial evidence toward class I status for INCB018424.
In human plasma after oral dosing with [14C]INCB018424, parent and identified metabolites accounted for 97% of the total radioactivity in circulation, indicating that there was a negligible amount of unidentified drug-related material. The primary metabolic pathways for INCB018424 in humans consisted of oxidation to single and multiple hydroxylated products as well as corresponding oxo metabolites, mainly occurring on the cyclopentyl moiety. A small percentage of these oxidative metabolites were subject further to O-glucuronidation. These results in humans were consistent with those of nonclinical species in which the route and extent of elimination were similarly rapid (≥80% of the dose excreted within 24 h after dosing) and unchanged drug constituted <20% of the radioactivity, indicating extensive metabolism (A. Shilling and F. Nedza, unpublished data).
The metabolite profiles between individuals were generally similar with the percentage of parent and the most abundant metabolite, M18 (2-hydroxycyclopentyl INCB018424), being consistent for the six subjects. Minor qualitative differences were observed among the stereoisomers of the 3-hydroxycyclopentyl and 3-oxocyclopentyl metabolites. In fact, the sum of parent and metabolites containing oxidation on the 2- or 3-position of the cyclopentyl moiety accounted for >90% of the circulating drug-related material. The clearance of metabolites appeared to be extended slightly compared with that for parent as indicated by the half-lives of radioactivity, although individual metabolites were undetectable by 24 h postdose, suggesting that accumulation of metabolites after multiple dosing is unlikely. This finding is supported by data from the multiple dose study in which the metabolite profiles were qualitatively and quantitatively similar on day 1 and day 10 with no apparent accumulation of parent or metabolites.
During the course of drug development, it is critical to demonstrate that the exposure of major metabolites in humans does not exceed that observed in nonclinical species during toxicology assessments (Baillie et al., 2002; Hastings et al., 2003). One metabolite, M18 (2-hydroxycyclopentyl INCB018424), constituted 17% of the radioactivity in plasma and would be defined as major in humans based on the current International Conference on Harmonization [M3(R2): Guidance on the Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals, 2009; http://www.ich.org/cache/compo/502-272-1.html#M3] (>10% of drug-related exposure). With use of the Food and Drug Administration Guidance for Industry: Safety Testing of Drug Metabolites, 2008; http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf), which defines a major human metabolite as 10% of parent exposure in plasma (based on AUC), two metabolites, M18 (30% of parent AUC) and a second peak containing two stereoisomers, M16 and M27 (3-hydroxycyclopentyl INCB018424), which constituted 8.7% of the drug-related material in human plasma (but plasma AUC was 14% of parent), would be considered major. This difference can be critical because toxicological evaluation of a human metabolite(s) is only warranted when a metabolite(s) meets this criterion and is observed at greater levels in humans than in animals in toxicity studies. The corresponding exposures (AUC) of these two metabolites in circulation after therapeutic doses of INCB018424 will be compared with plasma concentrations from oral studies in species used in toxicology studies at doses equal to or below the no-adverse-effect levels. These data will be essential for assessing whether metabolite exposures in animals approach human exposure, which would alleviate the need for further toxicity testing of the individual metabolite(s).
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033787.
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ABBREVIATIONS:
- JAK
- Janus tyrosine kinase
- INCB018424
- 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile
- HPLC
- high-performance liquid chromatography
- SPE
- solid-phase extraction
- LSC
- liquid scintillation counting
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- MRM
- multiple reaction monitoring
- AUC
- area under the plasma concentration-time curve.
- Received April 2, 2010.
- Accepted August 10, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics